The overall objective of this research is to extend our knowledge of structure-function relationships in thiamine diphosphate dependent a-keto acid decarboxylases, a large and ubiquitous class of enzymes of critical importance to metabolism and its associated energy production required for cellular function. The research focuses on enzymes that operate within the large, pyruvate dehydrogenase multienzyme complex (PDHc). Unlike the vast majority of biochemical reaction pathways that operate through simple diffusion of substrates and products between enzymes, PDHc and other multienzyme complexes employ substrate channeling between the E1, E2, and E3 enzymatic components. This provides a means of obtaining very high efficiency, and many key details are lacking regarding the required intramolecular interactions and processes. The E1 component from the E. coli PDHc complex is a member of the structurally underrepresented bacterial a2 E1 family, is thiamin diphosphate dependent, and is rate determining in the overall enzymatic reaction. It is also highly homologous in sequence with its counterparts in many other pathogenic organisms. The broad, long-term objective is to determine, analyze and understand the structure and function of an intact PDHc complex. The immediate objective is to exploit and build upon the structural and biochemical information obtained in the previous period for the E1 and E3 components from E. coli PDHc. Specific aim (1) is to provide detailed information about key protein-protein interactions necessary to assemble the functional multienzyme complex, and about the substrate channeling mechanism used to transfer products/substrates between enzymatic components within it. This will be achieved by determining and analyzing crystal structures of binary complexes made up from PDHc enzymatic components and/or their key fragments, and correlating them with the overall biological function. Specific aim (2) is to probe features associated with conformational changes previously found or thought to be necessary for stabilization of reaction intermediates and possibly protein-protein assembly, and to study the effects of protein-ligand interactions. To do this we will determine and analyze E1 structures and the associated protein-protein complexes in the presence of substrate, substrate analogs, and a new catalytic site directed inhibitor, as well as with some mutations introduced. Specific aim (3) is to probe mechanistic issues by examining structural ramifications arising from single residue mutations both in the active site, and along a proposed "proton wire" connecting active sites. For all aims x-ray crystallographic studies of isolated proteins, protein-ligand complexes, or protein-protein complexes will be coupled with biochemical data to obtain a complete picture of the process. Achieving these aims will help resolve the outstanding issues in thiamin-dependent enzymatic catalysis, and is the next step towards the long range goal of high resolution analysis of the entire 4.57 x 106 Dalton, 3 component 60 subunit containing PDHc complex. PUBLIC HEALTH RELEVANCE: The importance of thiamin catalyzed reactions has long been recognized, since abnormalities in either the availability of the vitamin B1 derived thiamin diphosphate or in the enzymes that utilize it have severe pathological consequences: for example, beri-beri, maple syrup urine disease, Pyruvate Dehydrogenase Deficiency (PDHA) associated lactic acidosis, microcephaly, motor neuropathy, Leigh syndrome, and neurological diseases including Alzheimer's and Parkinson's. The proposed work will advance our understanding of the precise mechanisms in key enzymatic pathways utilizing thiamin to provide energy for cellular functions, and should provide information likely to be useful in development of antibacterial agents that specifically target potent, pathogenic bacteria.